During the past 2.5 years with support from the current grant we have begun the breeding to produce congenic strains with the insulin 1 (N4 generation) and insulin 2 knockouts (N7generation) on the NOD background, and utilizing the speed congenic methodology have fixed NOD diabetes susceptibility regions. Constructs for the production of altered insulin sequences with retained biologic activity have been produced, and transgenic with germline transmission demonstuted (In collaboration with John Elliott). Our long term goal is to combine the insulin 1 and 2 knockouts with transgenes encoding altered insulin B:9-23 sequences in the same NOD strain and evaluate whether the B:9-23 native sequence is essential for the development of type 1 diabetes in the NOD mouse. Over the past two years we have continued our studies of autoimmunity directed at insulin with findings that include: 1. NOD T cell clones recognizing insulin peptide B:9-23 recognize two different overlapping insulin peptides, B:9-16 or B:13-23. 2. With a modified fluid phase radioassay insulin autoantibodies are readily detected in NOD mice and early expression of such autoantibodies correlate with early development of diabetes. 3. The insulin peptide B:9-23, specifically (no other insulin or proinsulin peptide tested) when administered to NOD mice induces insulin autoantibodies in an MHC restricted fashion (I-Ag7) or I-Ad) recognizing intact insulin concomitant with protection from diabetes. 4. The B:9-23 peptide, as well as B:13-23 when administered to normal Balb/C mice but not C57/B16 mice induces autoantibodies reacting with intact insulin that cannot be absorbed with the peptides but can be absorbed with "mouse" insulin. 5. Three different anti-islet transgenic mice (BDC2.5, Santamaria 4.1 and 8.3) all produce anti-insulin autoantibodies. 6. Insulitis can be induced in Balb/c mice by combining B:9-23 with poly1C, and diabetes can be induced in islet B7-1, H-2d/H-2b mice given B:19.-23 and poly-1C. During this same time period Wong and coworkers have characterized a CDS clone reacting with insulin peptide B:15-23 and have provided us with the clone and protocols for tetramer analysis for such T cells. We propose to: 1. Analyze congenic strains of NOD mice carrying the insulin 1 and 2 knockouts relative to expression of insulin autoantibodies, insulin peptide reactive T cells, and development of diabetes. 2. Produce strains of transgenic NOD mice with altered sequences of the B:9-23 insulin sequence 3. Evaluate NOD mice lacking native insulin 1 and 2 but with the transgene altered insulin for expression of insulin autoantibodies, insulin peptide reactive T cells, and development of diabetes. 4. Evaluate insulin peptide B:9-23 and altered peptide ligand for disease prevention in native NOD mice and mice with altered sequence.